New GcrR--Deficient Streptococcus Mutans Mutant For The Replacement Therapy Of Dental Caries

Dental caries are major oral health problem in most countries, affecting60-90%of schoolchildren and the vast majority of adults. According to the third national survey on oral health epidemiology in China,66.0%and28.9%of children at age5and12were affected by dental caries, meanwhile88.1%and98.4%of adults at35-44and65-74. Although fluoride and other preventive methods have led to decline in dental caries, little strategy puts effort on the actual pathogen for infection because the oral cavity is a complex ecosystem in which a rich and diverse micro biota has evolved. Methods such as probiotic approach (i.e. whole bacteria replacement therapy) are being investigated in order to eliminate pathogenic members of oral cavity.Mutans Streptococi is recognized as a main pathogen of dental caries in humans. In the procession of caries, the paramount virulence factor is the ability to produce acid.The sucrose-dependent adherence of S.mutans to teeth surface helps the bacteria not to be washed away with chewing or the flow of saliva. Interestingly, the rationale of the sucrose-dependent adherence could be used for the low acid-producing strains to enhance their adherence ability, so as to them to occupy the same ecological niche in plaque like their more cariogenic progenitor does.In the present study, we generated a gcrR-deficient S. mutans mutant and compared the morphological characteristics and the ability of adhesion and acid-production between the mutant and wild-type strains. It demonstrated that the mutant strain that is deficient in the WHOLE ORF of gcrR gene showed both lower acid production and stronger colonization potential in comparison with the wild-type strain.PART1Construction of an S.mutans gcrR-knockout mutantFirst of all, according to the gcrR gene sequence and its upstream and downstream homologous genes from the Genebank, the primers were designed. The resulting amplicon (DNA of S.mutans UA159as templates and gcrR-For/gcrR-Rev as primers) was a2118bp fragment including gcrR upstream (720bp) and gcrR downstream (705bp), which was then purified with a PCR Purification Kit (Qiagen, Netherlands), and ligated to the plasmid pMD18-T simple, according to the manufactures’instructions. The resultant plasmid pMD-G was used as a template in another round of amplification with the gcrR-rp-For/gcrR-rp-Rev primers set to generate gcrR-deficient ligation. The amplification product was then ligated to795bp of Kanamycin Resistance cassette (from pEGFP-N1vector) in the presence of T4DNA ligase (New England Biolabs, USA) to generate a new plasmid pMD-GK.Primers were designed by using Primer premier5.0software (Premier, Canada) and synthesized by Sangon Biotech Co.,Ltd (Shanghai, China). PCR amplicons were generated in a50μl final reaction volume with ExTaq mixture (Takara, Japan) using Primers gcrR-For/gcrR-Rev, gcrR-rp-For/gcrR-rp-Rev, and KanR-For/KanR-Rev. The reaction mixtures were placed in an automated thermal (Applied Biosystems, USA) that was programmed to run one cycle of denaturation at95℃for2min followed by40cycles of denaturation at95℃for5sec, primers annealing at60℃for30sec.To PMD-GK as a template for PCR with gcrR-For gcrR-Rev do a1%agarose gel electrophoresis, and approximately2.1kb at a specific band. Cleave the recombinant to Cla I and Xho I the plasmid pMD-GK by1%agarose gel electrophoresis, release the Kana resistant fragment of about795.1%agarose gel electrophoresis to the BamH I and Sal I cleave the recombinant plasmid after release2.2kb fragment. Will be sent to Shanghai Sangon sequencing, get downstream fragment and gcrR the Kana resistant fragment are100%correct. Since pMD18plasmid replication in E. coli, Streptococcus usually cannot be copied, and also has a resistance gene marker, is a more convenient to obtain the suicide vector can be used for homologous recombination. Homologous recombination is the positioning of the exogenous gene import receptor on the cell chromosome, since the sequence homology with the introduced gene, by a single or double-swap new gene fragment can replace a defective gene fragment in the seat, to modify the purpose of the gene.S.mutans UA159cells were transformed with the final plasmid pMD-GK in the presence of competence-stimulating peptide, as described elsewhere. Transformed colonies were selected on TH-agar plated with1mg/ml Kanamycin after overnight incubation at37℃with5%CO2.Kanamycin-resistant transformants were screened for deletion of the whole gcrR gene and replacement with Kanamycin-resistant cassette as a result of double-crossover recombination. Chromosomal DNA was isolated from selected transformants and from the UA159progenitor and used as a template for PCR amplification with primers P1/P2and RT-gcrRF/RT-gcrRR. The resulting amplicons (the gcrR upstream-KanR cassette junction and the part of gcrR gene) were analyzed by agarose gel electrophoresis and their sizes were compared with those predicted following a successful allelic exchange event. Nucleotide sequencing of the purified amplicons with the same primers confirmed the knockout of gcrR and insertion of the Kanamycin resistant cassette. The resulting mutant which contains the gcrR upstream-KanR cassette junction and lacks the gcrR gene was designated as MS-gcrR-def.PART2Biochemical and morphology study of the MS-gcrR-defS.mutans wild-type and the mutant MS-gcrR-def was grown in BHI supplemented with2%sucrose at37℃under5%CO2to mid-exponential-phase culture (OD470=0.5), then harvested by centrifugation at8000g and digested by N-acetylmuramidase (mutanolysinl; Sigma-Aldrich, USA) at20μg/ml and lysozyme (Sigma-Aldrich, USA) at10μg/ml at37℃for60min before homogenized in Trizol reagent (Invitrogen, USA). Following isopropanol precipitation, the RNA pellets were washed in70%ethanol and finally resuspended in nuclease-free water. RNA integrity was assessed by DuRed staining of the23S and16S rRNA subunits on a2%agarose gel, and quantified spectrophotometrically at A260for concentration and at A260/280for purity.RT-PCR was conducted to characterize the transcription of S.mutans gcrR gene. Briefly,1μg of DNase-treated total RNA isolated from S.mutans wild-type or the mutant MS-gcrR-def was reverse-transcripted to cDNA by using a first-strand cDNA synthesis kit (TaKaRa) in accordance with the recommendation of the supplier.Single-strand cDNA obtained in the above experiment, was amplified for gtfD and gbpC in a TaKaRa SYBR Premix system. Amplification was performed in a7500real-time PCR system (Applied Biosystems, USA) according to the following thermal cycling protocol:95℃for2min for initial denaturation, followed by40cycles of three steps consisting of94℃for5s, the optimal temperature (Table2) for60s, and72℃for60s. Quantification of gtfD and gbpC cDNA was normalized to the amount of cDNA derived from a DNA gyrase, subunit A protein (gyrA) control, the expression of which does not vary under the experimental conditions.The results of real-time RT-PCR revealed gtfD expression was increased in the MS-gcrR-def (mean RQ=3.8497for MS-gcrR-def,0.719for UA159and1.4487for MS-gcrR-def-com; n=3) relative to that in the wild type or compensated mutant. Likewise, gbpC expression was increased in MS-gcrR-def compared to that in the wild type or compensated mutants (mean RQ=19.9497for MS-gcrR-def,0.8317for UA159and1.0913 for MS-gcrR-def-com; n=3). Approximately, there were a fivefold and twenty-fourfold increase in gtfD and gbpC expression in the MS-gcrR-def gcrR knockout mutant relative to the wild type and compensated mutant.S.mutans wild-type and mutant strains were monitored for adherence to the walls of24well cell culture plates following2h,4h and6h-growths in BHI supplemented with2%sucrose. Briefly, overnight cultures of S.mutans UA159, the mutants MS-gcrR-def and MS-gcrR-def-com were inoculated as described above into BHI with control water and then standardized respectively to with1.0OD units (OD470) with fresh physiological saline. The three resulting bacteria suspensions were diluted1:20with fresh BHI with2%sucrose respectively and distributed into2.0ml fresh medium in the wells of sterile24-well cell culture plates in triplicate. The plate were kept at37℃with5%CO2for2h,4h and6h in triplicate. Bacterial growth was measured at470nm using a microplate reader. Then the media was discarded and the plates were rinsed with distilled water for3times and dried in room temperature.0.1%Crystal violet dye was added into the plates and kept for15min. Then the plates were rinsed with distilled water for3times and dried in room temperature or incubator. Thirty percent of acetic acid was added into plate to dissolve crystal violet. Biofilm formation was measured at562nm using a microplate reader.According to the result of adhesion test, the adhesion ability of gcrR deficient mutants was much stronger than that of the wild type. Furthermore, the difference in the early stage was greater than in the late stage.The two bacteria suspensions were diluted1:20with fresh BHI respectively or together and distributed4.0ml aliquots into the wells of sterile6-well cell culture plates to obtain two-bacterial biofilm cells at37℃for4h,6h,8h,11h and24h. In the6-well plates, each well was gently rinsed with distilled water to remove loose bacteria, and firmly attached cells were scraped from the substrate by pipetting and rinsed with distilled water to make sure all cells were collected. Total DNA was extracted from the same numbers of cells of the control and test groups.According to the result of Real time PCR, the mutant possessed an apparent advantage over wild type. The gcrR defective-bacteria accounted for more than90%in all adherent bacteria. Interestingly, the initial advantage continued to expand over time.In vitro acid production, the initial pH of the culture when mixed with the cell suspensions of S.mutans, MS-gcrR-def or MS-gcrR-def-com was measured at a constant pH of7.3and then the fall in pH over a16h period were measured again by pH meter to obtain the ApH of these cultures. the final pH of the above two cultures were significantly lower than those values for the culture of MS-gcrR-def which were in the range of4.11-4.13after16h of incubation at37℃.The MS-gcrR-def had a small average ApH of between3.17-3.19which was substantially smaller than the average ApH of the wild-type and MS-gcrR-def-com which ranged between3.23-3.25.There were significant differences between UA159and MS-gcrR-def (p<0.05). There were significant differences between MS-gcrR-def-com and MS-gcrR-def (p<0.05).After incubation at37℃with5%CO2, S.mutans UA159, MS-gcrR-def were harvested at mid-exponential phage (OD470=0.5) and the culture densities were standardized to1.0OD units with fresh physiological saline. The resulting cells suspensions were diluted by1:20into fresh BHI and distributed2.0ml aliquots into the wells of24-well plates in triplicate. The cells were incubated for24h at37℃and5%CO2in and OD470measurements were obtained with gentle agitation preceding each reading in a Microplate Reader. Doubling times from each of three independent experiments were determined for cells in exponential phase and at points corresponding.We observed equal survivor-ship for all S.mutans strain following24h of growth. MS-gcrR-def ultimately attained a lower final resting culture density than wild-type. Taken together, these findings support the involvement of GcrR in the ability of S.mutans to withstand acid stress and indicate that defects in acid tolerance arise from gcrR deficiency.The cells suspensions were smeared on glass slides stained with Gram’s iodine and observed under an optical microscope. The results of the wild type and gcrR-deficient mutant stain were obviously Gram positive. Interestingly, the mutants were more easily clump together than the wild type and the process occurred rapidly. It contributed to bacterial colonization in early stage.The biofilm samples were gently rinsed with PBS three times to remove unattached cells, dried for5min, and then stained with molecular Probes SYTO9and propdium iodide (Invitrogen, USA). The stained samples were then examined by CLSM (ZEISS LSM510META; Carl Zeiss, Germany). The red and green fluorescence intensity was quantified respectively by Image pro-plus6.0(Media Cybernetics, USA).According to the figures, at the early stage (at2h and4h) MS-gcrR-def formed biofilms more quickly than UA159and the biofilms were thicker relative to the wild type.It demonstrated that MS-gcrR-def showed both lower acid production and stronger colonization potential in comparison with the wild-type strain. Part3Assessment of the cariogenic potential of MS-gcrR-def in SD rats.After feeding with appropriate antibiotic for3days, two groups of six rats each were challenged with oral swabs saturated with2X109CFU/ml of S.mutans UA159and MS-gcrR-def per ml. Over the next3day, the rats were challenged repeatedly with fresh swabs of the appropriate strain, and on the next day postchallenge, swab samples were collected and plated onto MSB agar plated to confirm colonization. The rats of MS-gcrR-def group were provided water supplemented with1%the appropriate strain and maintained on caries-promoting diet2000#. Forty days postchallenge, the rats were sacrificed, and their maxillary and mandibles were removed and subsequently processed for caries scores.MS-gcrR-def was significantly less cariogenic than the UA159wild-type progenitor in germfree rats.